The introduction of biologically active molecules, such as for example DNA, RNA or proteins, into living cells is an important tool for the analysis of biological functions of these molecules. A preferred method for the introduction of foreign molecules into cells is electroporation which, in contrast to chemical methods, does not depend on the simultaneous transport of other biologically active molecules. In electroporation, the foreign molecules are introduced into the cells from a buffer solution adapted to the cells or from a cell culture medium via a brief current flow. The cell membrane is being made permeable to the foreign molecules by the action of the short electrical pulses. In addition, the cell suspension is frequently located in a so-called cuvette, i.e. a narrow vessel that is open at the top, and whose interior is formed by two pairs of side walls arranged parallel and opposite to one another.
The interior can receive the cell suspension, i.e. generally an aqueous buffer solution or a cell culture medium, in which the cells to be treated are suspended. Such cuvettes generally have a pair of electrodes in the lower region of a pair of opposing side walls, which allow for the application of an electric voltage. An electrical discharge at these electrodes results in an electrical current flowing between the electrodes and through the cell suspension, causing nucleic acids or other molecules to be transported into the cells or leading, depending on the conditions selected, to cell fusion.
As a result of the brief application of a strong electrical field, i.e. a short pulse with high current density, cells, cell derivatives, sub-cellular particles and/or vesicles can also be fused. In this so-called electrofusion, the cells are at first brought into close membrane contact, for example via an inhomogeneous electrical alternating field. The subsequent application of an electric field pulse results in the interaction of membrane parts which eventually leads to fusion. For electrofusion, apparatuses may be used which are comparable to those used for electroporation.
During electroporation, the biologically active molecules initially enter the cytoplasm through the temporarily produced ‘pores’ in the cell membrane. In certain cases, the molecules may already perform the function of interest in the cytoplasm and, subsequently, under certain conditions, may also enter the nucleus. In particular with applications in which the biologically active molecules can only carry out the function of interest in the nucleus, for example, if the expression of a gene is to be analysed, and, in particular, if cells without, or with only low, division rates are used, for example primary cells, it is advantageous if the biologically active molecules are transported directly into the nucleus.
It is known from the electroporation method disclosed in US2004014220, which is incorporated herein by reference in its entirety, that in such cases, to achieve high transfection efficiency, a strong electrical field having a field strength of at least 2 kV/cm has to be generated in the buffer solution for a preset duration of at least 10 μs via a high voltage pulse.
A method for treating biological material by means of high electrical currents is also known from US2005064596, which is incorporated herein by reference in its entirety. In the method disclosed therein, the biological material is added to a buffer solution having an ionic strength of at least 200 mmol/l to ensure a low cell mortality rate while accomplishing high transfection efficiency.
Primarily in biochemical and pharmaceutical applications, in which a plurality of reaction batches have to be tested simultaneously and in the shortest possible time, in particular in HT analyses (HT=high throughput), it is necessary to provide as large a number of reaction chambers as possible, for example 96 or 384. The reaction vessels used in this context are generally referred to as multi well plates, microtitration plates or multi wells. The individual reaction chambers (‘wells’) of these vessels are relatively small and can therefore only receive small volumes. Moreover, it is frequently advantageous to use smaller sample volumes to save buffer and cell material. In addition, in particular with valuable cell material, for example primary cells, only small amounts of cells are generally available. It is therefore frequently desirable and in certain instances necessary to work with small sample volumes.
Electrical hydrolysis cannot be excluded as a side effect when generating strong electrical fields in liquids. In the mildest case, electrolysis can be noticed by the formation of gas bubbles on the surfaces of the electrodes, which in turn leads to the formation of foam. In an extreme case, explosion-type gas formation occurs, which due to the resulting displacement effect, leads to the expulsion of the samples from the area between the electrodes (referred to hereinafter as ‘spattering’). The latter generally results in the loss of sample(s) or at least in the sample not remaining in the electrical field for the time intended. The spattering of a sample therefore qualitatively and/or quantitatively impairs the result of a test or sample processing and moreover has a negative effect on the reproducibility of the results. Accordingly, in the various applications where treatment of biological cells in electrical fields is necessary, in particular during electroporation, electrolysis constitutes an undesirable side effect.
In theory, the probability of spattering could be reduced by reducing the electrical conductivity. Higher cells which are not provided with rigid cell walls, however, can generally only survive in solutions with a specific osmolarity. Generally, electrolytes are also amongst the osmotically effective dissolved substances which result in a more or less high electrical conductivity of the solution. For example, to carry out electroporation, it is generally necessary to introduce ions into the cell suspension and, as disclosed in US2005064596, which is incorporated herein by reference in its entirety, also advantageous. Thus, for practical reasons, there are limits to reducing the probability of spattering by reducing the electrical conductivity. Accordingly, in such cases electrolysis of varying degrees can be expected.
The occurrence of spattering is hereby a stochastic event. This means that the event can only be described by probabilities. Depending on the prescribed conditions, the frequency of undesired spattering may, for example, be under 5%, but can also be over 95%. The probability of spattering is, hereby particularly high when low volumes are used at a high current density. In order to develop a process to the production stage, the problem poses itself to reduce this probability by appropriate methods, which are to be employed by the customer to under 1%, for example.
Thus, there is a need in the art to provide a method of the aforementioned type in which the frequency of the undesired expulsion of a sample from the area between the electrodes is significantly reduced.